Our MS data were collected similar to as described previously38 (link). In brief, we used a Q Exactive mass spectrometer (Thermo Fisher Scientific, San Jose, CA) coupled with a Famos Autosampler (LC Packings) and an Accela600 LC pump (Thermo Fisher Scientific). Peptides were separated on a 100 μm inner diameter microcapillary column packed with ∼25 cm of Accucore C18 resin (2.6 μm, 150 Å, Thermo Fisher Scientific). For each analysis, we loaded ~1 μg onto the column.
Peptides were separated using a 1 h gradient of 5–25% acetonitrile in 0.125% formic acid with a flow rate of ∼300 nl/min. The scan sequence began with an Orbitrap MS1 spectrum with the following parameters: resolution 70,000, scan range 300–1500 Th, automatic gain control (AGC) target 1 × 105, maximum injection time 250 ms, and centroid spectrum data type. We selected the top 20 precursors for MS2 analysis which consisted of HCD high-energy collision dissociation with the following parameters: resolution 17,500, AGC 1 × 105, maximum injection time 60 ms, isolation window 2 Th, normalized collision energy 30, and centroid spectrum data type. The underfill ratio was set at 1%, which corresponds to a 1.1 × 104 intensity threshold. In addition, unassigned and singly charged species were excluded from MS2 analysis and dynamic exclusion was set to automatic.
Peptides were separated using a 1 h gradient of 5–25% acetonitrile in 0.125% formic acid with a flow rate of ∼300 nl/min. The scan sequence began with an Orbitrap MS1 spectrum with the following parameters: resolution 70,000, scan range 300–1500 Th, automatic gain control (AGC) target 1 × 105, maximum injection time 250 ms, and centroid spectrum data type. We selected the top 20 precursors for MS2 analysis which consisted of HCD high-energy collision dissociation with the following parameters: resolution 17,500, AGC 1 × 105, maximum injection time 60 ms, isolation window 2 Th, normalized collision energy 30, and centroid spectrum data type. The underfill ratio was set at 1%, which corresponds to a 1.1 × 104 intensity threshold. In addition, unassigned and singly charged species were excluded from MS2 analysis and dynamic exclusion was set to automatic.
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